ARTICLE Received 11 Jul 2012 | Accepted 7 Sep 2012 | Published 9 Oct 2012 DOI: 10.1038/ncomms2127 Orphan receptor IL-17RD tunes IL-17A signalling and is required for neutrophilia Mark Mellett1, Paola Atzei1, Alan Horgan1, Emily Hams2, Thomas Floss3, Wolfgang Wurst3, Padraic G. Fallon2,4,5 & Paul N. Moynagh1 Interleukin-17A, the prototypical member of the interleukin-17 cytokine family, coordinates local tissue inflammation by recruiting neutrophils to sites of infection. Dysregulation of interleukin-17 signalling has been linked to the pathogenesis of inflammatory diseases and autoimmunity. The interleukin-17 receptor family members (A–E) have a broad range of functional effects in immune signalling yet no known role has been described for the remaining orphan receptor, interleukin-17 receptor D, in regulating interleukin-17A-induced signalling pathways. Here we demonstrate that interleukin-17 receptor D can differentially regulate the various pathways employed by interleukin-17A. Neutrophil recruitment, in response to in vivo administration of interleukin-17A, is abolished in interleukin-17 receptor D-deficient mice, correlating with reduced interleukin-17A-induced activation of p38 mitogen-activated protein kinase and expression of the neutrophil chemokine MIP-2. In contrast, interleukin-17 receptor D deficiency results in enhanced interleukin-17A-induced activation of nuclear factor-kappa B and interleukin-6 and keratinocyte chemoattractant expression. Interleukin-17 receptor D disrupts the interaction of Act1 and TRAF6 causing differential regulation of nuclear factor- kappa B and p38 mitogen-activated protein kinase signalling pathways. 1 Department of Biology, Institute of Immunology, National University of Ireland Maynooth, Maynooth, Ireland. 2 Institute of Molecular Medicine, School of Medicine, Trinity College Dublin, Dublin 8, Ireland. 3 Helmholtz Zentrum München, Institute of Developmental Genetics, 85764 Munich, Germany. 4 National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland. 5 Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland. Correspondence and requests for materials should be addressed to P.N.M. (email: [email protected]). NATURE COMMUNICATIONS | 3:1119 | DOI: 10.1038/ncomms2127 | www.nature.com/naturecommunications © 2012 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2127 ro-inflammatory cytokines of the interleukin-17 (IL-17) luciferase reporter plasmid and IL-17RD-specific small interfering family are secreted by cells of the activated immune system, RNA (siRNA) or a scrambled control siRNA. The IL-17RD-specific Pincluding TH17 cells, γδ-T cells and natural killer cells, and siRNA strongly suppressed IL-17RD expression whereas the contribute to host defence against extracellular pathogens1. IL-17 control siRNA was without effect (Fig. 1a). Knockdown of IL-17RD signalling pathways are also implicated in the pathogenesis of auto- augmented the ability of human IL-17A to induce the expression immune and inflammatory diseases, and IL-17 is found in increased of NF-κB-regulated luciferase (Fig. 1a). The control siRNA was concentrations in psoriasis, rheumatoid arthritis, inflammatory without effect confirming the specificity of the effects of IL-17RD bowel disease and the lungs of patients with allergic asthma2–5. siRNA. Additionally, IL-17A-induced expression of the NF-κB- The IL-17 cytokine family consists of six ligands (IL-17A-F), responsive gene IL-6 was enhanced in IL-17RD-knockdown cells which signal through five receptors (IL-17RA-E). IL-17 receptors (Fig. 1b), consistent with an inhibitory role for IL-17RD in regulating are widely expressed bestowing the cytokines with broad effector IL-17A-induced activation of NF-κB. This was further confirmed functions6,7. To date, IL-17RA and IL-17RC have been the best- by overexpressed IL-17RD inhibiting IL-17A-induced activation of characterized members of the IL-17 receptor family, and IL-17RA can NF-κB activation in HeLa cells (Fig. 1c). form heterodimers with IL-17RC in initiating immune responses to To further validate these effects, we established U373 cell lines extracellular pathogens8. IL-17A and IL-17F can form homodimers transduced with lentivirus to stably express IL-17RD-specific short- or heterodimers and IL-17RA- and IL-17RC-deficient mice show hairpin RNA (shRNA) or control shRNA. Knockdown efficiency was impaired responsiveness to both ligands9,10. IL-17RA binds to regularly checked in these U373-shRNA stable cell lines and 40–60% IL-17A with a higher affinity than IL-17F in humans, whereas the knockdown of Il17rd mRNA was consistently achieved compared converse is true of IL-17RC11. Murine IL-17RA can bind both with the control-shRNA (Fig. 1d). The knockdown of IL-17RD in IL-17A and IL-17F but murine IL-17RC can only bind IL-17F. these cells resulted in augmented IL-17A-induced phosphorylation Recent findings have revealed a new role for IL-17RE in intestinal of IκB and upstream IKKs (Fig. 1e) and this was associated with immunity in response to bacterial infection, with IL-17RE forming enhanced NF-κB–DNA binding (Fig. 1f). Again, knockdown of IL- a receptor complex with IL-17RA to bind IL-17C12–14. 17RD in this model resulted in an enhancement of IL-17A-induced IL-17A coordinates tissue inflammation and mobilizes neu- expression of IL-6 (Fig. 1g). To further validate that augmented IL-6 trophils by stimulating the production of cytokines and chemokines, production in IL-17RD-knockdown cells is directly attributable to including IL-6, keratinocyte chemoattractant (KC) and macrophage diminished expression of IL-17RD, we demonstrated that the expres- inflammatory protein-2 (MIP-2) in mice, and IL-8 and growth- sion of exogenous IL-17RD strongly suppressed IL-17A induction of regulated oncogene family members in humans15–18. IL-17RA regu- IL-6 in cells stably expressing IL-17RD shRNA (Fig. 1h). lates gene expression by activating nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways18. Mem- IL-17RD differentially regulates IL-17A-responsive genes. The bers of the IL-17 receptor family share an intracellular Sef/IL-17R above knockdown studies suggested IL-17RD to be a negative regu- (SEFIR) domain that is important for triggering downstream sig- lator of IL-17A signalling. To confirm the physiological relevance of nalling19. Stimulation of IL-17RA leads to an association with the such a role, it was necessary to generate IL-17RD-deficient mice and adaptor Act1, via SEFIR–SEFIR interactions, followed by recruit- this was achieved by gene-trap technology. The gene that encodes ment and polyubiquitination of TRAF6, thus initiating downstream murine IL-17RD consists of 13 exons. The targeting vector disrupted activation of NF-κB and MAPKs20,21. Recent findings have also the Il17rd gene between the first and second exons with aβ -geomy- detailed a messenger RNA (mRNA)-stability pathway bifurcating cin-resistance gene cassette (Supplementary Fig. S1a). Il17rd − / − downstream of Act1 that is independent of TRAF6 (ref. 22). pups were born in an expected Mendelian ratio and showed no obvi- Although recent studies have delineated important immunomod- ous phenotypic abnormalities and this is consistent with previously ulatory roles for the various IL-17R members, IL-17 receptor D generated IL-17RD-deficient mice25. As expected, Il17rd mRNA (IL-17RD) remains an orphan receptor with no reported physi- expression is abolished in Il17rd − / − mice and replaced by β-Geo ological role in IL-17 signalling, apart from a study in which over- expression (Supplementary Fig. S1b). Quantitative PCR was used expression of IL-17RD was shown to co-operate with IL-17RA23. to confirm disruption of the gene encoding IL-17RD in Il17rd − / − IL-17RD was originally termed similar expression to fgf (Sef) due murine embryonic fibroblasts (MEFs) (Supplementary Fig. S1c). its coexpression with fgf genes in zebrafish embryo development24. Il17rd − / − MEFs were used to assess a potential regulatory role for Here, we describe for the first time an important physiological IL-17RD by measuring IL-6 expression in response to principal role for IL-17RD in IL-17 signalling. IL-17RD negatively regulates IL-17RA cytokines, IL-17A and IL-17F (Fig. 2a). IL-17A was the IL-17A-induced activation of NF-κB and the expression of more efficacious inducer of IL-6 expression from MEFs from wild- pro-inflammatory genes such as IL-6. IL-17RD coordinates these type mice and its efficacy was greatly enhanced inIl17rd − / − MEFS. regulatory effects by negatively regulating Act1/TRAF6 binding A more detailed characterization of the regulatory effects of IL-17RD and disrupting the IL-17RA/Act1/TRAF6 signalling axis. However, on IL-17A signalling was then performed. This initially involved an conversely IL-17RD promotes IL-17A-induced activation of p38 assessment of the effects of IL-17RD deficiency on the expression MAPK and induction of MIP-2. In vivo, the loss of IL-17A-induced of a number of IL-17A-responsive genes in MEFs. Similar to IL-6, MIP-2 expression in IL-17RD-deficient mice is associated with loss of IL-17RD resulted in significantly enhanced KC expression in diminished IL-17A-induced neutrophil infiltration in the lungs response to IL-17A (Fig. 2b). In contrast IL-17A-induced expres- and the peritoneum, whereas exogenous MIP-2 delivery restores sion of the chemokine MIP-2 was diminished in Il17rd − / − MEFs, neutrophilia in these mice. From these findings it is evident that indicating that IL-17RD can differentially regulate the expression IL-17RD differentially regulates IL-17A-induced NF-κB and MAPK of IL-17A-responsive genes. This was independent of cell type as pri- signalling pathways, and is an important mediator in facilitating mary bone marrow-derived macrophages (BMDMs) from Il17rd − / − IL-17A-induced neutrophilia. mice also displayed enhanced expression of IL-6 and KC but reduced expression of MIP-2 in response to IL-17A (Fig.